pH-sensitive fluorescent dyes employed in biological research and medical diagnostics belong to two groups, each distinguished by the origin of fluorescent responses to changes in pH. The first group includes compounds having fluorescence controlled by the ionization of phenolic hydroxyl groups in a fluorophore. Examples include fluorescein, carboxyfluorescein, Oregon Green®, SNARF®, SNAFL®, and HPTS indicators.
U.S. Patent Publication No. 2006/0051874 describes fluorescein-like structures incorporated into a fluorescent detector for monitoring pH of the blood in bank storages. Because the degree of ionization of these types of molecules increases upon lowering the acidity of the environment, they become more fluorescent as pH increases.
Fluorescent pH sensors of the second group include an amino group (aliphatic or aromatic) as an indicator moiety along with a reporter fluorescent dye moiety. When such a molecule absorbs a photon creating an excited electronic state, the electron of the amino group's unshared pair transfers to the orbital vacated by excitation. Such an electron transfer, referred to as Photoinduced Electron Transfer (PET) prevents the excited molecule from emission transition, thus the fluorescence of the dye is quenched. Protonation of the amino group changes the nature and energy of the pair's orbital and stops the PET. As a result, the fluorescent reporter moiety responds to a pH change. Because protonation of the amino group cancels the quenching, the PET-based sensors become more fluorescent as pH decreases.
Examples of PET-based pH sensors include LysoSensor™ dyes, which contain a dimethylamino group as an indicator moiety and CypHer® 5E dye which has an indolenine indicator group. One disadvantage of these sensors is that the working range is shifted to the acidic side because of the low pKa of the indicator amino group.
A family of rhodamine-based pH sensors is described in PCT Publication No. WO 2005/098437 (Smith et al.). The dyes have a benzene ring substituted ortho to the xanthene moiety by —OH or —SH (or their deprotonated forms), such that deprotonation to a negatively charged state quenches the fluorescence and it is only upon protonation of the negatively charged —O− or —S− to a neutral state that the fluorescence is restored. Typically, the pH at which this occurs is less than pH 6. WO 2005/098437 purports that the ionized state of the —OH or —SH group is responsible for the pH response of the dye and that the strong electron withdrawing properties of the tetramethylrhodamine moiety in the dyes significantly decreases the pKa of the indicator group, thus shifting the sensors' working range toward highly acidic pH values. However, this limits the applicability of the dyes described in WO 2005/098437 at a physiological pH (e.g., pH 6-7), especially in biological systems. An additional disadvantage of these dyes is that their pKa is not tunable. Furthermore, these compounds have been found by us to be unstable in solution.
Accordingly, there is a need for additional pH-sensitive fluorescent dyes with improved properties, including in at least some compounds the ability to detect pH changes in biological systems. It is an object of the present invention to develop a novel class of relatively stable fluorescent pH sensors that fluoresce in the red portion of the UV/VIS spectrum, preferably with a working range towards neutral and other biologically relevant pH values that mitigate or remove the disadvantages of the compounds known in art.